Encoding NF-kappaB temporal control in response to TNF: distinct roles for the negative regulators IkappaBalpha and A20

Abstract

TNF-induced NF-kappaB activity shows complex temporal regulation whose different phases lead to distinct gene expression programs. Combining experimental studies and mathematical modeling, we identify two temporal amplification steps-one determined by the obligate negative feedback regulator IkappaBalpha-that define the duration of the first phase of NF-kappaB activity. The second phase is defined by A20, whose inducible expression provides for a rheostat function by which other inflammatory stimuli can regulate TNF responses. Our results delineate the nonredundant functions implied by the knockout phenotypes of ikappabalpha and a20, and identify the latter as a signaling cross-talk mediator controlling inflammatory and developmental responses.

Figure 1.

A mathematical model of TNFR signaling to NF-κB. (A) Schematic depicting TNF signaling from TNFR to IKK, which functions as the input to the NF-κB signaling module. The two most rapidly NF-κB-inducible attenuators, IκBα and A20, are shown. A detailed schematic of the model is available in the Supplemental Material. (B) Computational simulations of persistent TNF stimulation depicting free TNF levels and the activities of IKK and NF-κB. (C) Computational simulations of IKK (top) and NF-κB (bottom) activities in response to 1-, 2-, 5-, or 15-min TNF pulses. (D) IKK and NF-κB activities were experimentally measured in wild-type MEFs in response to 1-, 2-, 5-, or 15-min 1 ng/mL TNF pulses via in vitro IP-kinase assay and EMSA, respectively. (E) RPA to track expression of NF-κB target genes in wild-type MEFs stimulated with a 1-min pulse of TNF (1 ng/mL).

Figure 2.

Inducible expression is critical for the function of IκBα but not A20. (A) Simulations of nuclear NF-κB activity in wild-type, iκbα^−/−, and a20^−/− MEFs in response to TNF. The data are presented as graphs (top) and as heat maps (below) in which the level of NF-κB is color-coded from 0 nM (blue) to 100 nM (red). (B) Simulations of nuclear NF-κB activity in models with altered constitutive transcription rates of IκBα (left) or A20 (right) in the absence of inducible transcription. In each simulation the constitutive transcription rate was multiplied by one of 21 constitutive transcriptional modifiers, ranging from 2⁻¹⁰, 2⁻⁹, 2⁻⁸. . .1. . .2⁸, 2⁹, 2¹⁰. The results were graphed over time (X-axis), with the value of the rate modifier on the Y-axis. NF-κB activity is color-coded as in Figure 2A. (C) Simulations of nuclear NF-κB activity in models possessing both constitutive and inducible expression of IκBα (wild type), or each individually. Nuclear NF-κB activity was then measured via EMSA in wild-type cells, or in iκbα^−/− cells reconstituted with a constitutively expressing iκbα transgene (pBABE.IκBα.puro) or an empty vector control (pBABE.EV.puro, labeled iκbα^−/−) in response to a 15-min pulse of TNF (1 ng/mL). (D) Simulations predict NF-κB activity in TNF-treated cells that have either constitutive or inducible A20 expression, or both (wild type). Nuclear NF-κB activity was measured via EMSA in wild-type cells, or in a20^−/− cells reconstituted with a constitutively expressing a20 transgene (pBABE.A20.puro), an NF-κB-inducible transgene (fIL8.A20.puro), or an empty vector control (pBABE.EV.puro, labeled a20^−/−) in response to persistent TNF stimulation (1 ng/mL).

Figure 3.

A20 can mediate signaling cross-talk between inflammatory stimuli. (A) Quantitated A20 and IκBα mRNA expression in MEFs stimulated with 1 ng/mL TNF or IL-1, as measured by RPA. (B) Nuclear NF-κB activity was measured via EMSA in wild-type, a20^−/−, and iκbα^−/− cells in response to 1 ng/mL TNF or IL-1 stimulation. (C) Simulation of TNF–NF-κB dose response in naïve (black) and IL-1 pretreated (blue) cells. Computational simulations calculated the maximal nuclear NF-κB activity for TNF doses ranging from 10⁻³ to 10³ ng/mL. IL-1 pretreatment was simulated as a 1-h stimulation followed by 1 h of “rest” prior to TNF stimulation. (D) Nuclear NF-κB activity in response to persistent TNF stimulation (0.1 or 1 ng/mL) was measured by EMSA in wild-type and a20^−/− cells that were naïve (−) or pretreated with IL-1 for 1 h, followed by 1 h or 24 h of “rest.”

Figure 4.

Temporal dose response analysis of TNF-induced NF-κB activity. (A) Simulation of NF-κB activity in wild-type, iκbα^−/−, and a20^−/− cells in response to TNF pulses ranging from 1 min to 180 min in 1-min increments. The results were graphed over time (hours), with the pulse duration (hours) on the Y-axis. NF-κB activity (nanomolar) was color-coded as in Figure 2. (B) NF-κB activity profiles were simulated in response to 5-, 15-, or 45-min TNF pulses in wild-type and a20^−/− cells (top), and were then measured experimentally via EMSA (bottom). (C) Schematic summary of how negative feedback regulators IκBα and A20 encode NF-κB activity dynamics in the TNF signaling pathway. Whereas dynamic feedback (yellow box) is critical to IκBα’s function, inducible expression of A20 confers a tunable rheostat (blue box) function. Via this rheostat function, A20 mediates signaling cross-talk, for example, from prior cellular exposure to IL-1. (Below) TNF produces a typically biphasic NF-κB activity that is encoded by the differential functions of A20 and IκBα. The duration of the first phase is a function of the inducibility (change in synthesis rate, or second-order derivative, denoted by “^..”) of IκBα, but is not a function of the TNF stimulus duration or concentration. The duration of the second phase is a function of the concentration of the A20 protein at that time. High concentrations of A20 protein during the early phase (as a result of prior NF-κB activity) may also affect the amplitude of the first phase, but not its duration.